Obesity-linked insulin resistance and type 2 diabetes are intimately linked to adipocyte dysfunction, increased adipocyte lipolysis, and lipid accretion in tissues other than adipose. In obesity, the hypertrophied adipocyte is not able to properiy store excess fatty acids, the rate of lipolysis is increased, and these lipids deposit in other tissues where they hamper insulin action. Inhibiting obesity-linked adipocyte lipolysis can improve insulin sensitivity. All enzymes involved in adipocyte lipolysis belong to the serine hydrolase family. Despite their importance in fat cell physiology, the majority of serine hydrolases have not been studied. Serine hydrolases (SHs) are a key enzyme family involved in metabolism and adipocyte function, v\/here they contribute to lipolysis, lipogenesis, and lipid uptake. Yet, more than 50% ofthe 120+ human serine hydrolases, including some that have been genetically linked to human disease, remain unannotated, have no known function or physiological substrates, and most lack inhibitors to aid in their characterization and therapeuti validation. Because individual SHs already constitute targets for drugs that treat metabolic disease, it is reasonable to hypothesize that important additional drug targets will be found among the numerous SHs that remain uncharacterized. Discerning which of these unannotated SHs are relevant in adipocyte function and which may serve as therapeutic targets for obesity-diabetes is a very complex problem. The critically important research challenge that this project addresses is the identification and therapeutic validation of pooriy annotated metabolic serine hydrolases that play key roles in adipocyte function. Our multidisciplinary team will achieve this goal by combining cutting-edge chemoproteomic and metabolomics methods with deep biological expertise in obesity and type 2 diabetes. Specifically, we intend to globally identify and assess the therapeutic potential of unannotated SHs active in adipocytes and whose activity is modulated in physiologic conditions and in obesity-diabetes. Some of these enzymes may be new targets for metabolic disease. In the process, we will create first-in-class chemical probes and genetic models to study adipocyte SHs that will be distributed to the larger research community.